Fungal infections are a growing medical threat, particularly for immunocompromised individuals, yet little is understood about the molecular basis of immune evasion by fungal pathogens. The long-term goal of this research is to understand how intracellular fungal pathogens subvert host defenses. This aim will fundamentally interface with the goal of this program project application, which is to understand how diverse pathogens such as Francisella tularensis, Listeria monocytogenes, and Mycobacterium tuberculosis manipulate macrophage responses. We hypothesize that a comparative analysis of macrophage responses to these pathogens, in addition to a fungal intracellular pathogen, is a more powerful means of dissecting host response than studying any single intracellular pathogen in isolation. Our model system is Histoplasma capsulatum, an intracellular fungal pathogen that survives and replicates in the phagosome of macrophages. How H. capsulatum colonizes a niche that is normally hostile to microbes is a mystery. We hypothesize that H. capsulatum produces gene products that inhibit acidification of the host phagosome, thereby allowing fungal cells to survive and grow intracellularly. The objective of this proposal is to use molecular genetic approaches to uncover which H. capsulatum molecules are required for intracellular survival and growth. Furthermore, functional genomics will be used to identify host genes and pathways that are targeted by H. capsulatum. Our specific aims are to (1) use well-established assays to characterize the stages of infection of murine bone-marrow derived macrophages with H. capsulatum; (2) perform a genetic screen to identify Histoplasma genes required for survival and growth in macrophages; and (3) compare the global gene expression profile of macrophages during infection with wild-type or mutant H. capsulatum and the other intracellular pathogens mentioned above. These studies will identify regulatory circuits in host cells that are manipulated by H. capsulatum and other key intracellular pathogens relevant to biodefense.